Plant biomass provides an abundant source of potential energy in the form of carbohydrates that can be used in numerous industrial and agricultural processes and, therefore, is an important renewable source for generating fermentable sugars. Fermentation of these sugars can produce valuable commercial end products such as biofuels and biochemicals.
Although fermentation of sugars to ethanol is relatively straightforward, efficient conversion of cellulosic biomass to fermentable sugars such as glucose is more challenging. The huge potential energy of large amounts of carbohydrates in plant biomass is not sufficiently used because the sugars form part of complex polymers (polysaccharides, such as cellulose and hemicellulose) and, therefore, are not easily accessible for fermentation. Thus, cellulose can be pre-treated mechanically, chemically, enzymatically or in other ways to increase its susceptibility to hydrolysis. After this pre-treatment process, there is a saccharification or hydrolysis stage consisting of an enzymatic process in which complex carbohydrates (such as starch or cellulose) are hydrolysed into their monosaccharide components. The goal of any saccharification technology therefore is to change or remove structural and compositional obstacles in order to improve the rate of enzymatic hydrolysis and increase the yield of fermentable sugars obtained from cellulose or hemicellulose (N. Mosier et al., 2005, Bioresource Technology 96, 673-686). After the saccharification stage, the fermentation process is performed. Therefore, the higher the amount of complex sugars remaining at the end of the hydrolytic process, the lower the yield in ethanol production at the end of the fermentation process. Thus, an area of research directed at reducing costs and improving the yield of biofuel production processes is focussed on improving the technical efficiency of hydrolytic enzymes, or generally on improving the efficiency of enzyme cocktails used to generate fermentable sugars from biomass.
It has been shown that individual enzymes are only capable of partially digesting cellulose and hemicellulose and therefore the combined action of different classes of enzymes is required to complete their conversion into monomeric sugars. Many more enzymes are required for digesting hemicellulose to monomeric sugars that for cellulose, including enzymes with xylanase, beta-xylosidase, arabinofuranosidase, mannanase, galactosidase and glucuronidase activity. Other enzymes without glycosyl hydrolase activity can also be involved such as acetyl xylan esterase and ferulic acid esterase. Therefore, enzymatic hydrolysis of polysaccharides for their conversion to soluble sugars and, finally, to monomers such as xylose, glucose and other pentoses and hexoses are catalysed by various enzymes that together are called “cellulases”. Cellulases are multienzyme complexes comprising at least three main components, endo-β-glucanase (EC 3.2.1.4), exo-β-glucanase or cellobiohydrolase (EC 3.2.1.9.1) and β-glucosidase (EC 3.2.1.21), and it has been shown that they act synergistically in the hydrolysis of cellulose (Woodward, J. 1991, Bioresource Technology Vol 36, pp. 67-75).
Microbial cellulases have become focal biocatalysts because of their complex nature and their extensive industrial applications (Kuhad R. C. et al., 2011, Enzyme Research, Article ID 280696). Recently, considerable attention has been paid to current knowledge on the production of cellulases and the challenges in cellulases researching have been focus especially in obtaining cellulases with higher activity and improved properties.
On the other hand, glycosyl hydrolase proteins of family 61 (GH61) have been known for over 20 years. These GH61 proteins are accessory proteins that contribute to cellulose degradation. The fact that these enzymes act by direct oxidation of cellulose, rather than by hydrolysis, has led to their current name: Cu dependent polysaccharide monooxygenases (Polysaccharide Monooxygenase; PMOs). Compared to other cellulolytic enzymes, PMOs are relatively small proteins with typical molecular weights of between 20 and 50 kDa (Baldrian and Valaskova 2008, FEMS Microbiology Reviews 32: 501-521; Harris et al., 2010, Biochemistry 49: 3305-3316). These proteins require two oxygen molecules to cause product breakdown and oxidation. One of these molecules derives from water, the other enters the reaction in the form of molecular oxygen, which is necessary for direct oxidation of the substrate. Therefore, members of this enzyme family act as Cu monooxygenases that catalyse the breakdown of cellulose by an oxidative mechanism, releasing cellodextrins (Langston et al., 2011, Applied and Environmental Microbiology 77: 7007-7015).
The hydrolytic efficiency of a multi-enzyme complex in the saccharification process of cellulosic material depends both on the properties of the individual enzymes and on the proportion of each enzyme present in the complex. Therefore, in the context of biofuel production processes, enzyme cocktails need to be designed with improved individual activities. Specifically, it would be an advantage in the art to improve the activity and stability of PMO polypeptides. In this sense several publications have proposed the supplementation of the enzymatic mixtures containing cellulases and PMOs with copper, which is a cofactor of PMOs, to increase the activity and stability of these enzymes (US2014127771, WO2012138772).
In summary, the use of enzymatic mixtures containing PMO polypeptides with improved activity and/or stability during the saccharification or hydrolysis stage of cellulosic biomass will lead to an improvement in the yield of this stage through an increase in the amount of final fermentable sugars. Later, these sugars can be fermented to produce biofuels such as bioethanol, so this would ultimately increase the efficiency and profitability of the whole biofuel production process.